Many common bacterial pathogens can delay virulence factor production until there are a sufficient number of cells such that, working together, the group can overwhelm a host's defenses. To coordinate such an attack, some species use a method of cell-cell communication called quorum sensing (QS) (Camilli and Bassler, 2006; Rutherford and Bassler, 2012). In Gram-negative bacteria, QS involves the production of a membrane diffusible small molecule signal, often an N-acyl L-homoserine lactone (AHL), that accumulates in the surroundings at a concentration proportional to cell density (Fuqua and Greenberg, 2002). At a threshold concentration, this signal is bound by, and activates, an intracellular LuxR-type receptor that acts as a transcriptional regulator to induce the expression of group-beneficial genes.
QS systems are often induced in response to environmental signals (Hense and Schuster, 2015, Lee and Zhang, 2015). Such mechanisms allow the bacterium to delay the energetically costly production of QS signals and virulence factors until it is in an environment permissive to infection. Because of their association with virulence, QS systems are considered to be potential antivirulence targets (Cegelski et al., 2008, Allen et al., 2014, Gerdt and Blackwell, 2014).
A number of research groups are actively developing small molecule and macromolecular agents capable of inhibiting QS receptor activity (Galloway et al., 2011, Murray et al., 2014, Amara et al., 2011, Praneenararat et al., 2012).
The opportunistic pathogen Pseudomonas aeruginosa is highly adaptable to life in a variety of environments ranging from soil to water to skin. P. aeruginosa possesses a sophisticated QS system that incorporates a large degree of environmental regulation (Wagner et al., 2003, Duan and Surette, 2007, Williams and Cámara, 2009). P. aeruginosa has three QS systems—Las, Rhl, and Pqs (FIGS. 2A and 2B)—whose associated LuxR-type receptors (LasR and RhlR) and LysR type receptor PqsR; (also known as MvfR) regulate distinct subsets of virulence-associated genes upon activation by their cognate small molecule signal (Venturi, 2006, Schuster and Greenberg, 2008). Using these systems, P. aeruginosa is able to colonize a variety of mammalian tissues including, notoriously, the airways of patients suffering from cystic fibrosis (CF) (Lyczak et al., 2000, Folkesson et al., 2012). In the canonical model of P. aeruginosa QS, it has been understood that a regulatory hierarchy exists between the three QS systems, whereby Las induces the expression and activation of both Rhl and Pqs, while an inverse regulatory relationship exists between the latter systems (Balasubramanian et al., 2013).
Increasing evidence has revealed that nutritional cues found in infection environments can alter this hierarchy (Dekimpe and Déziel, 2009; Cabeen, 2014; Lee and Zhang, 2015). For example, cellular factors that sense low levels of iron and phosphate can directly stimulate the Rhl and Pqs systems, bypassing Las (FIG. 2B) (Jensen et al., 2006; Oglesby et al., 2008; Lee et al., 2013). In addition, the chemical nature and availability of carbon sources can suppress or induce specific QS systems via the downstream effects of carbon catabolite repression and the stringent response (FIG. 1A) (Shrout et al., 2006; Schafhauser et al., 2014; Yang et al., 2015). A plausible explanation for the existence of the complex QS network in P. aeruginosa is that it serves to tune the virulence profile of the organism in response to diverse environmental stimuli (Mellbye and Schuster, 2014).
Despite considerable recent research, a full understanding of the mechanisms by which Las, Rhl, and Pqs work together to accomplish this regulation remains elusive. Mellbye and Schuster have shown that Las-responsive genes are primarily induced in a cell density-dependent manner, while Rhl-associated genes are up-regulated in response to environmental cues (Mellbye and Schuster, 2014). Although Pqs is an important regulator of global virulence, how it fits into this model is unknown (Déziel et al., 2005; Rampioni et al., 2010). The inverse regulation between Rhl and Pqs suggests a close relationship; yet, the relative contribution of Rhl and Pqs to virulence factor production is poorly defined. Further, whether Las remains important for virulence factor production in wild-type (WT) P. aeruginosa under conditions that directly stimulate Rhl and Pqs is unclear. Thus, there is a need in the art for a more complete understanding of how these systems work together to coordinate virulence, particularly in defined, infection-relevant environments.